Expression of human estrogen receptors in transgenic mice

ABSTRACT

The present invention relates to improved compositions and methods for assaying the efficacy of novel drugs functioning either as agonists or antagonists at nuclear receptors. In particular, the invention provides transgenic non-human animals comprising a human steroid hormone receptor gene operably linked to a promoter which directs expression of the steroid hormone receptor gene in an epithelial cell of the non-human animals.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. CA71358,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention pertains to the field of transgenic non-human animals. Inparticular this invention pertains to transgenic animals that expresshuman steroid hormone receptor genes in desired tissues, such as theepithelium, and to methods of screening potential therapeutics foractivity at steroid hormone receptors.

BACKGROUND OF THE INVENTION

Steroid hormones are secreted by the adrenal cortex, testis, ovary andplacenta and include the androgens (such as testosterone), estrogens(such as estradiol and estrone), glucocorticoids (cortisone,corticosterone, and cortisol), mineralocorticoids (primarilyaldosterone), and progestogens (primarily progesterone). Steroidhormones regulate proliferation and differentiation in target cellswithin the reproductive tract, mammary gland, and peripheral tissuessuch as the bones, heart, blood vessels, and hair follicles (for areview, see Yamashita et al., Localization and functions of steroidhormone receptors, 1998, Histol. Histopathol. 13(1):255-70).

Steroid hormones are highly lipophilic and act through nuclear receptorsrather than through receptors on the plasma membrane. Steroid hormonereceptors have been shown to support the development of cancer in thebreast, prostate, uterus, cervix, and ovaries. In addition, steroidhormone receptors appear to prevent osteoporosis of the bones, toprevent atherosclerosis of the coronary arteries of the heart, and tomediate baldness in men. Therefore, steroid hormone nuclear receptorsand other gene products that are involved in steroid hormone metabolismare attractive targets for the development of therapeutics that addressthe treatment of reproductive cancers and conditions such asosteoporosis, atherosclerosis, and baldness.

Steroid hormone receptors are part of a family of nuclear receptorswhich contain a hormone-biding region and a DNA-binding region, andthereby act as transcription enhancers. Upon binding to their specificligands, nuclear receptors interact directly with regions of DNA inorder to influence transcription of genes regulating hormonal activity.See, Ribeiro et al., The nuclear hormone receptor gene superfamily,1995, Annual Rev Med. 46:443-53. There is a growing list of drugs thateither bind directly to steroid hormone receptors or modulate steroidhormone metabolism. Drugs that bind directly to the nuclear receptorsinclude tamoxifen, an anti-estrogen used in treatment of breast cancer,and raloxifene, used in prevention of osteoporosis. Drugs that modulatesex steroid metabolism include aromatase inhibitors, used to treatbreast cancer and possibly prostate cancer, and finasteride (Propecia™),used in the treatment of hair loss.

Models of targeted expression of oncogenes and growth factors to theepidermis of transgenic mice have been described. These models have usedkeratin promoters to target the expression of foreign DNA. The basalcell specific keratin-14 (K14) promoter has been used to express growthfactors and oncogenes in transgenic animals as models for thedevelopment of specific carcinomas (Vassar et al., 1991, Cell64:365-380; U.S. Pat. No. 5,698,764). There is currently no transgenicmodel for the expression of human nuclear receptors in the epithelium.

Despite the existence of several drugs that either bind directly tosteroid hormone receptors or modulate steroid hormone metabolism, thereis a further need to develop models for assaying and testing the site ofaction and the efficacy of drugs that act to modulate nuclear receptoractivity and metabolism. The present invention addresses these and otherneeds.

SUMMARY OF THE INVENTION

The present invention provides transgenic non-human animals exhibiting adetectable phenotype, typically epidermal hyperplasia, caused by asteroid hormone receptor gene operably linked to a promoter whichdirects expression of the steroid receptor gene in the epithelium in thenon-human animal, preferably a mouse. The steroid receptor gene ispreferably a human estrogen receptor gene. The promoter used to driveexpression of the steroid receptor gene is preferably a keratin-14promoter directing expression of the steroid receptor in a basalkeratinocyte.

The animal may further comprise a β-galactosidase gene operably linkedto a promoter which directs expression of the β-galactosidase gene in anepithelial cell. If the steroid hormone receptor gene is expressed in abasal keratinocyte, the β-galactosidase gene is also preferablyexpressed in a basal keratinocyte.

The invention also provides DNA constructs comprising an expressioncassette including a steroid hormone receptor gene operably linked to apromoter which directs expression of the steroid hormone receptor in theepithelium, preferably in a basal keratinocyte. The promoter ispreferably the keratin-14 promoter.

The invention further provides methods of testing a composition for theability to modulate steroid hormone receptor activity. The methodscomprise providing a transgenic non-human animal comprising a humansteroid hormone receptor gene operably linked to a keratin-14 promoterwhich directs expression of the gene in an epithelial cell,administering the composition to the non-human animal, and detectingchanges in the epithelial cell of the non-human animal.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, “non-human animals” include, for example, mammals suchas non-human primates, ovine, canine, bovine, rattus and murine species,as well as rabbits and the like. Preferred non-human animals areselected from the rodent family, including rat, guinea pig and mouse,most preferably mouse.

As used herein, a “steroid” or “steroid hormone” refers to any one of agroup of biologically active compounds synthesized from cholesterol thatcontain a cyclopentanoperhydrophenanthrene nucleus. A “steroid receptor”or “steroid hormone receptor” is a nuclear receptor that binds a steroidor steroid hormone described herein.

As used herein, the term “steroid hormone receptor gene” or “steroidreceptor gene” refers to a nucleotide sequence, or any subsequencethereof, that encodes a steroid receptor described herein or thatencodes a gene product exhibiting DNA-binding and steroidhormone-binding activity, in vitro or in vivo; and any conservativelymodified variants thereof. Also explicitly included within thisdefinition are both wild-type and mutant genes (e.g. mutant steroidhormone receptor genes isolated from cancer cells) that may or may nothave altered activity as compared to wild-type genes.

The term “estrogen receptor” refers to a known nuclear receptor having apredicted molecular weight of about 66-kd, that is activated byestrogenic steroid hormones such as estradiol. The active form of theprotein enhances expression of genes involved in the formation ofsecondary sexual characteristics in mammalian females. An estrogenreceptor can be an allele, polymorphic variant, interspecies homolog, orany subsequence thereof that exhibits estrogenic steroid hormone-bindingactivity.

As used herein, “estrogen receptor gene” is a wild-type or mutantnucleotide sequence that encodes an estrogen receptor described herein,and conservatively modified variants thereof. An example of an estrogenreceptor gene is described in Greene et al., 1986, Science231(4742):1150-4. One of ordinary skill in the art will recognize thatcertain modifications, additions, and deletions may be made to theestrogen receptor gene sequence which will not affect the function oractivity of the gene product. Such variants are included within thisdefinition. An example of a mutant estrogen receptor gene is K206A, inwhich the lysine at position 206 is replaced by alanine.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence. For example, apromoter is operably linked to a coding sequence if it controlstranscription of the sequence.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

An “expression cassette” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression cassette can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

A “detectable phenotype” is any symptom or characteristic of an organismwhich is measurable by any one or a number of different objectivecriteria, including, but not limited to, visual inspection, microscopicobservation, antibody labeling, DNA labeling, or assays for changes ingene expression which are known in the art. Detectable phenotypes mayinclude, but are not limited to, skin thickening, redness, flaking,epidermal hyperplasia, dysplasia, altered nuclear labeling, or alteredprotein or gene expression.

The term “modulate the activity” means to have an effect on, e.g., toincrease or inhibit or otherwise alter, the activity of, e.g., a humannuclear receptor gene.

The term “administering” the composition means contacting by anyconventional method known to one of skill in the art, such as, forexample, parenteral, oral, topical, and inhalation routes.

The phrase “detecting changes” refers to using one or a number ofdifferent objective criteria, including, but not limited to, visualinspection, microscopic observation, antibody labeling, DNA labeling, orassays for changes in gene expression which are known in the art, todetermine the effect of a composition on a non-human animal of theinvention.

“Recombinant” refers to a human manipulated polynucleotide or a copy orcomplement of a human manipulated polynucleotide. For instance, arecombinant expression cassette comprising a promoter operably linked toa second polynucleotide may include a promoter that is heterologous tothe second polynucleotide as the result of human manipulation (e.g., bymethods described in Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)or Current Protocols in Molecular Biology Volumes 1-3, John Wiley &Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising theexpression cassette. In another example, a recombinant expressioncassette may comprise polynucleotides combined in such a way that thepolynucleotides are extremely unlikely to be found in nature. Forinstance, human manipulated restriction sites or plasmid vectorsequences may flank or separate the promoter from the secondpolynucleotide. One of skill will recognize that polynucleotides can bemanipulated in many ways and are not limited to the examples above.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to sequences or subsequences that have atleast 60%, preferably 70%, more preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity when aligned for maximumcorrespondence over a comparison window as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection: This definition also refers to the complement of atest sequence, which has substantial sequence or subsequencecomplementarity when the test sequence has substantial identity to areference sequence.

One of skill in the art will recognize that two polypeptides can also be“substantially identical” if the two polypeptides are immunologicallysimilar. Thus, overall protein structure may be similar while theprimary structure of the two polypeptides display significant variation.Therefore a method to measure whether two polypeptides are substantiallyidentical involves measuring the binding of monoclonal or polyclonalantibodies to each polypeptide. Two polypeptides are substantiallyidentical if the antibodies specific for a first polypeptide bind to asecond polypeptide with an affinity of at least one third of theaffinity for the first polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff& Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, in a nucleic acid, peptide, polypeptide, or proteinsequence which alters a single amino acid or a small percentage of aminoacids in the encoded sequence is a “conservatively modified variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid. Thus,a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two molecules or theircomplements hybridize to each other under stringent conditions, asdescribed below.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, highly stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength pH. Lower stringency conditions aregenerally selected to be about 15-30° C. below the T_(m). The T_(m) isthe temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 time background hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.

In the present invention, genomic DNA or cDNA comprising nucleic acidsof the invention can be identified in standard Southern blots understringent conditions using the nucleic acid sequences disclosed here.For the purposes of this disclosure, suitable stringent conditions forsuch hybridizations are those which include a hybridization in a bufferof 40% formamide, 1 M NaCl, 1% SDS at 37° C., and at least one wash in0.2×SSC at a temperature of at least about 50° C., usually about 55° C.to about 60° C., for 20 minutes, or equivalent conditions. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar stringency.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers, can then be used as a probe under stringenthybridization conditions to isolate the test sequence from a cDNA orgenomic library, or to identify the test sequence in, e.g., an RNA gelor DNA gel blot hybridization analysis.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

I. Introduction

Steroid hormones are associated with the development of breast,prostate, uterine, cervical, and ovarian cancer. In addition, steroidhormones mediate baldness in men, and are important in the prevention ofosteoporosis, and atherosclerosis of the coronary arteries of the heart.Steroid hormone activity is mediated by nuclear steroid hormonereceptors. Therefore, steroid hormone receptors and other proteinsinvolved in steroid hormone metabolism are attractive targets for thedevelopment and testing of therapeutics that address the treatment ofreproductive cancers and conditions such as osteoporosis,atherosclerosis, and baldness.

The present invention provides transgenic animals and DNA constructs fortargeting steroid hormone receptor genes to the epithelium of transgenicanimals which exhibit an easily observable phenotype. Alternatively, thereceptor genes can be targeted to the various tissues in which thesecancers occur (e.g. breast, prostate, uterine, cervical and ovariantissues). These animals, e.g., mice, provide a convenient assay toevaluate the effect of compositions that function either as agonists orantagonists of the target steroid hormone receptors and metabolizingenzymes. The present invention is thus useful in the testing ofpotential therapeutic drugs that target either steroid hormone receptorsor other genes that are involved in the metabolism of steroid hormones,e.g, genes encoding enzymes such as aromatase. For example a testcompound can first be evaluated in a transgenic animal in which thesteroid hormone receptor gene is targeted to the epithelium and thentested further in an animal in which the gene is targeted to a desiredtissue, e.g. breast, in the case of screens for compounds useful intreating breast cancer.

Generally, the nomenclature used hereafter and the laboratory proceduresin molecular genetics described below are those well known and commonlyemployed in the art. Standard techniques are used for recombinantnucleic acid methods, polynucleotide synthesis, cell culture, andtransgene incorporation (e.g., electroporation, microinjection,lipofection). Generally enzymatic reactions, oligonucleotide synthesis,and purification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various general andspecific references which are provided throughout this document. Theprocedures therein are believed to be well known in the art and areprovided for the convenience of the reader. Much of the nomenclature andgeneral laboratory procedures described below can be found in Sambrook,et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

II. DNA Constructs

Appropriate constructs for production of vectors used to make transgenicanimals are described in Hogan et al., 1986, Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. In theconstruction of vectors for the production of transgenic animals, thecoding sequence of interest is typically operably linked to expressionregulatory sequences. In such transgenes, the expression regulatorysequence is at least the minimal sequences required for efficientcell-type specific expression, which generally are at least a promoterand at least about 1 kilobase (kb) upstream of the promoter. Usually thesequences upstream of the promoter are used contiguously, althoughvarious deletions and rearrangements can be employed. Some desiredregulatory elements (e.g., enhancers, silencers) may be relativelyposition-insensitive, so that the regulatory element will functioncorrectly even if positioned differently in a transgene than in thecorresponding germline gene. For example, an enhancer may be located ata different distance from a promoter, in a different orientation, and/orin a different linear order. For example, an enhancer that is located 3′to a promoter in germline configuration might be located 5′ to thepromoter in a transgene.

Typically, expression regulation sequences are chosen to producetissue-specific or cell type-specific expression of the desiredstructural gene. In the present invention the targeted cells are, e.g.,breast, prostate, uterine, cervical, ovarian or epithelial cells. Once atissue or cell type is chosen for expression, expression regulationsequences are chosen. Generally, such expression regulation sequencesare derived from genes that are expressed primarily in the tissue orcell type chosen. Preferably, the genes from which these expressionregulation sequences are obtained are expressed substantially only inthe tissue or cell type chosen, although secondary expression in othertissue and/or cell types is acceptable if expression of the recombinantDNA in the transgene in such tissue or cell type is not detrimental tothe transgenic animal.

The constructs will usually also comprise downstream expressionregulation sequences to supplement tissue or cell-type specificexpression. The downstream expression regulation sequences includepolyadenylation sequences (either from the endogenous gene or from othersources such as SV40) and sequences that may affect RNA stability aswell as enhancer and/or other sequences which enhance expression.

In some embodiments of the present invention, a steroid hormone receptorgene is placed in an expression cassette under the control of a promoterthat will direct expression of the gene to an epithelial cell. A numberof suitable promoters can be used to direct expression of the steroidhormone nuclear receptor gene in epithelial cells. Particularly usefulfor targeting the expression of sequences to epithelial cells are thepromoters from genes encoding keratin. Keratins are proteins that areexpressed in epithelial tissues. Specific keratin proteins, identifiedby a number, e.g., keratin-5, are exclusively expressed not only incertain epithelia, but also in selected cells populating the epithelia.The epidermis is composed of layers of cells (keratinocytes) whichproduce specific types of keratin proteins. The basal cells producekeratin 5 and 14 (K5 and K14), whereas the more mature, terminallydifferentiated keratinocytes, e.g., the suprabasal keratinocytes,produce K10 and K1. Promoters from other keratin genes, such as K8 andK19 are useful in directing expression to epithelia in the bladder orintestines.

In a preferred embodiment, a basal cell keratin promoter (e.g., K5 orK14) is utilized. The K14 promoter is particularly preferred. A K14expression cassette, containing 2 kb of the K14 promoter/enhancer and500 bp of the 3′ flanking sequence including the K14 polyadenylationsignal, has been shown to appropriately target expression of transgenesto the basal cells of squameous epithelium (Vassar et al., 1991, Cell64:365-380; Cheng et al., 1992 Genes Dev. 6:1444-1456; Guo et al., 1993,EMBO J. 12:973-986; Turksen et al., 1992, Proc. Natl. Acad. Sci. USA89:5068-5072; Vassar et al., 1989, Proc. Natl. Acad. Sci. USA86:1563-1567; U.S. Pat. No. 5,698,764). This cassette is preferred forconstruction of the transgene of the present invention.

Alternatively, the steroid hormone receptor genes can be targeted toother tissues, such as breast, prostate, uterine, cervical, and ovariantissue. Promoters for expression in these tissues are known to those ofskill. Examples of suitable promoters include the mouse mammary tumorvirus (MMTV) promoter (see, e.g. Guy et al. Mol. Cell. Biol. 12:954-961(1992)) for expression in breast tissue and the probasin promoter forexpression in prostate cells (see, e.g. Yan et al. Prostate 32:129-130(1997))/

In preferred embodiments, steroid hormone receptor genes are used in theDNA constructs of the invention. One of skill will recognize that thegenes need not be naturally occurring wild-type genes, but can beconservatively modified variants or mutants. The human estrogen receptor(ER) gene is conveniently used. See Greene et al., Sequence andexpression of human estrogen receptor complementary DNA, 1986, Science231(4742):1150-4. One mutant ER gene that can be used is the K206Amutant, in which the lysine at position 206 is substituted with alanine.One of skill in the art will recognize that any other steroid hormonereceptor gene can be substituted. For example, the androgen,progesterone, mineralocorticoid, and glucocorticoid receptor genes canconveniently be substituted. See, e.g., Lubahn et al., The humanandrogen receptor: complementary deoxyribonucleic acid cloning, sequenceanalysis and gene expression in prostate, 1988, Mol Endocrinol.2(12):1265-75; Misrahi et al., Complete amino acid sequence of the humanprogesterone receptor deduced from cloned cDNA, 1987, Biochem BiophysRes Commun. 143(2):740-8; Arriza et al., Cloning of humanmineralocorticoid receptor complementary DNA: structural and functionalkinship with the glucocorticoid receptor, 1987, Science237(4812):268-75; Govindan et al., Cloning of the human glucocorticoidreceptor cDNA, 1985, Nucleic Acids Res. 13(23):8293-304.

Further, genes encoding metabolizing enzymes that regulate steroidhormones are suitable targets for the DNA constructs and transgenicanimals of the invention. For example, the gene encoding aromatase, anenzyme that converts androgens to estrogen, is conveniently substituted.See, Harada et al., Cloning of a complete cDNA encoding human aromatase:immunochemical identification and sequence analysis, 1988, BiochemBiophys Res Commun. 156(2):725-32. Aromatase inhibitors are used totreat breast cancer patients. The recent discovery of a distinct genefor another estrogen receptor (estrogen receptor-beta) suggests thatprostate cancer may respond to aromatase inhibitors as well. Chang etal., Estrogen receptor-beta: implications for the prostate gland, 1999,Prostate 40(2):115-24.

Methods of modifying the DNA constructs of the invention are well knownto those of skill in the art (see, for example, Sambrook et al., 1989supra; Methods in Enzymology, 1987, Vol. 152: Guide to Molecular CloningTechniques, Berger and Kimmel, eds., San Diego, Academic Press, Inc.; orAusubel et al., 1987, Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, N.Y.).

III. Production of Transgenic Animals

The transgenic non-human animals of the invention are produced byintroducing expression cassettes comprising the desired promoted andstructural gene (e.g. K14-estrogen receptor transgene) into the germlineof the non-human animal. In a preferred embodiment, the transgenicnon-human animal is selected from the rodent family, including rat,guinea pig and mouse. Most preferably, the transgenic animal is a mouse.The transgenic animal may also be a non-human primate, a member of theovine, canine, or bovine species, or a rabbit and the like.

In preferred embodiments two expression cassettes are introduced intothe animal. One cassette comprises the recombinant expression cassetteof the invention and the other comprises a selectable marker gene suchas β-galactosidase or β-glucuronidase (GUS), useful in monitoringexpression of the transgene. Genes encoding β-galactosidase areparticularly preferred. Embryonic target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonic target cell.

A. MicroInjection Methods

Microinjection is a preferred method for transforming a zygote or earlystage embryo. In the mouse, the male pronucleus reaches the size ofapproximately 20 micrometers in diameter which allows reproducibleinjection of 1-2 pl of DNA solution. The use of zygotes as a target forgene transfer has a major advantage in that in most cases the injectedDNA will be incorporated into the host gene before the first cleavage(Brinster, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 4438-4442). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will, in general, also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

The gene sequence being introduced need not be incorporated into aself-replicating plasmid or virus (Jaenisch, 1988, Science, 240:1468-1474). However, in a preferred embodiment, the gene sequence willbe introduced in a as a cassette comprising the gene under the controlof a promoter. The promoter acts to regulate transcription of the genein response to endogenous factors present in a particular tissue andthus results in tissue-specific expression of the gene.

Once the DNA molecule has been injected into the fertilized egg cell,the cell is implanted into the uterus of a recipient female, and allowedto develop into an animal. Since all of the animal's cells are derivedfrom the implanted fertilized egg, all of the cells of the resultinganimal (including the germ line cells) shall contain the introduced genesequence. If, as occurs in about 30% of events, the first cellulardivision occurs before the introduced gene sequence has integrated intothe cell's genome, the resulting animal will be a chimeric animal.

By breeding and inbreeding such animals, it has been possible to produceheterozygous and homozygous transgenic animals. Despite anyunpredictability in the formation of such transgenic animals, theanimals have generally been found to be stable, and to be capable ofproducing offspring which retain and express the introduced genesequence.

The success rate for producing transgenic animals is greatest in mice.Approximately 25% of fertilized mouse eggs into which DNA has beeninjected, and which have been implanted in a female, will becometransgenic mice. A number of other transgenic animals have also beenproduced. These include rabbits, sheep, cattle, and pigs (Jaenisch,1988, Science 240: 1468-1474; Hammer et al., 1986, J. Animal Sci, 63:269; Hammer et al., 1985, Nature 315: 680; Wagner et al., 1984Theriogenology 21: 29).

B. Retroviral Methods

Retroviral infection can also be used to introduce a transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, 1976, Proc. Natl. Acad. SciUSA 73: 1260-1264). Efficient infection of the blastomeres is obtainedby enzymatic treatment to remove the zona pellucida (Hogan, et al.,1986, In Manipulating the Mouse Embryo, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). The viral vector system used tointroduce the transgene is typically a replication-defective retroviruscarrying the transgene (Jahner, et al., 1985, Proc. Natl. Acad. Sci. USA82, 6927-6931; Van der Putten, et al., 1985, Proc. Natl. Acad. Sci. USA82, 6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra ; Stewart et al., 1987, EMBO J., 6: 383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal., 1982, Nature, 298: 623-628). Most of the founders will be mosaicfor the transgene since incorporation occurs only in a subset of thecells which formed the transgenic non-human animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retroviral infectionof the midgestation embryo (Jahner et al., 1982, supra).

C. ES Cell Implantation

A third target cell for transgene introduction is the embryonic stemcell (ES). ES cells are obtained from pre-implantation embryos culturedin vitro (Evans et al., 1981, Nature, 292: 154-156; Bradley, et al.,1984, Nature, 309: 255-258; Gossler, et al., 1986, Proc. Natl. Acad. SciUSA 83:, 9065-9069; and Robertson, et al., 1986, Nature, 322: 445-448).Transgenes can be efficiently introduced into ES cells using a number ofmeans well known to those of skill in the art. Such transformed ES cellscan thereafter be combined with blastocysts from a non-human animal. TheES cells thereafter colonize the embryo and contribute to the germ lineof the resulting chimeric animal (for a review see Jaenisch, 1988,Science, 240: 1468-1474).

In a preferred embodiment, the DNA is introduced by electroporation(Toneguzzo et al., 1988, Nucleic Acids Res., 16: 5515-5532; Quillet etal., 1988, J. Immunol., 141: 17-20; Machy et al., 1988, Proc. Nat'l.Acad. Sci. USA, 85: 8027-8031). After permitting the introduction of theDNA molecule(s), the cells are cultured under conventional conditions,as are known in the art.

In order to facilitate the recovery of those cells which have receivedthe DNA molecule containing the desired gene sequence, it is preferableto introduce the DNA containing the desired gene sequence in combinationwith a second gene sequence which would contain a detectable marker genesequence. For the purposes of the present invention, any gene sequencewhose presence in a cell permits one to recognize and clonally isolatethe cell may be employed as a detectable (selectable) marker genesequence. The presence of the detectable (selectable) marker sequence ina recipient cell may be recognized by PCR, by detection of radiolabellednucleotides, or by other assays of detection which do not require theexpression of the detectable marker sequence. Typically, the detectablemarker gene sequence will be expressed in the recipient cell, and willresult in a selectable phenotype. Selectable markers are well known tothose of skill in the art. Some examples include the hprt gene(Littlefield, 1964, Science 145: 709-710), the tk (thymidine kinase)gene of herpes simplex virus (Giphart-Gassler et al., 1989, Mutat, Res.,214: 223-232), the nDtII gene (Thomas et al., 1987, Cell, 51: 503-512;Mansour et al., 1988, Nature 336: 348-352), or other genes which conferresistance to amino acid or nucleoside analogues, or antibiotics, etc.

Any ES cell may be used in accordance with the present invention. It is,however, preferred to use primary isolates of ES cells. Such isolatesmay be obtained directly from embryos such as the CCE cell linedisclosed by Robertson, E. J., 1989, In: Current Communications inMolecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, ColdSpring Harbor, N.Y., pp. 39-44), or from the clonal isolation of EScells from the CCE cell line (Schwartzberg et al., 1989, Science 212:799-803). Such clonal isolation may be accomplished according to themethod of Robertson, 1987, In: Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, Ed., IRL Press, Oxford.The purpose of such clonal propagation is to obtain ES cells which havea greater efficiency for differentiating into an animal. Clonallyselected ES cells are approximately 10-fold more effective in producingtransgenic animals than the progenitor cell line CCE.

D. Verification of the Presence of the Transgene in the Animal

A number of methods used to detect particular polynucleotide sequencescan be used to verify that the desired sequences have been integratedinto the genome of the transgenic animal. For instance, Fluorescent InSitu Hybridization (FISH) can be used to detect the transgene in tissuefrom the animal. Several guides to FISH techniques are available, e.g.,Gall et al,. 1981, Meth. Enzymol., 21:470-480, and Angerer et al., 1985,in Genetic Engineering: Principles and Methods Setlow and Hollaender,Eds. Vol 7, pg. 43-65. Alternatively, DNA or RNA can be isolated fortissue (typically tail tissue). The desired sequences can be detected bySouthern or Northern hybridization or by PCR using primers and probesspecific for the transgene. Standard PCR methods useful in the presentinvention are described in PCR Protocols: A Guide to Methods andApplications (Innis et al., eds., Academic Press, San Diego 1990).

Alternatively, expression of the integrated gene can also be detected bydetecting the gene product. The protein can be detected, for instance,using standard immunoblotting techniques, well known to those of skillin the art.

IV. Methods to Evaluate the Efficacy of Compositions Functioning atSteroid Hormone Receptors

A valuable feature of the transgenic animals of the invention is aneasily observable phenotype in the epidermis of the transgenic animal.In some embodiments, the non-human transgenic animals exhibit epidermalhyperplasia. They may alternatively, or in addition, exhibit otherphenotypes, including, but not limited to, thickening (acanthosis),redness (erythema), or flaking (hyperkeratosis). Therefore, thetransgenic animals of the invention are useful in methods for screeningdrugs and treatments designed to target nuclear steroid hormonereceptors and other proteins involved in steroid hormone metabolism.

The methods herein provided for testing a composition comprise providinga transgenic non-human animal comprising a human steroid hormonereceptor gene operably linked to a keratin-14 promoter, administeringthe composition to the non-human animal, and detecting changes in theepidermis of the non-human animal. The compositions that are tested bythis method will depend on the target transgene of the transgenicanimal. For example, the K14-estrogen receptor mouse of the inventioncan be used to assay the efficacy of anti-estrogen drugs, such astamoxifen, raloxifen, and the like. Similarly, a transgenic animalexpressing a K14-androgen receptor transgene in hair follicles can beused to assay drugs directed at androgen receptors for the treatment ofbaldness. Still further, a transgenic animal comprising a transgeneencoding an enzyme involved in steroid hormone metabolism can be used toassay drugs thought to modulate sex steroid metabolism. Examples of suchdrugs include aromatase inhibitors, and fenesteride (Propesia™), whichblocks an enzyme that converts testosterone into the related hormonedihydrotestosterone.

In a preferred embodiment, the epithelium of the epidermis of thetransgenic animal is used as a readout to detect changes in thetransgenic animal and evaluate the efficacy of compositions functioningeither as agonists or antagonists at nuclear receptors and otherproteins involved in steroid hormone metabolism. The advantage of usingthe epidermis as an assay is that it can be conveniently evaluated by anumber of different visual and microscopic objective criteria, as wellas by molecular assays. For example, the epidermis can be visuallyinspected for changes such as thickening (acanthosis), redness(erythema), and flaking (hyperkeratosis). In addition, the skin can beevaluated microscopically for hyperplastic thickening and alterations ofappearance which are consistent either with normal differentiation, orwith dysplasia, such as the persistence of immature neoplastic cells inthe upper epidermal layers. At the molecular level, panels of keratinantibodies exist which can identify the keratin expression patterns oftransgenic versus non-transgenic epidermis, as each layer of theepidermis produces a repertoire of keratin intermediate filamentsspecific for cell types within that layer. Still further, the DNAlabeling pattern of epidermis is distinct and can be used to compare thenumber of labeled nuclei from transgenic and non-transgenic epidermis.

V. EXAMPLES

K-14-ER Transgenic Mice

Standard techniques were used to microinject a DNA construct comprisingthe human estrogen receptor (ER) linked to the enhancer/promoter of thehuman keratin-14 (K14) gene into B6D2/F2 mouse embryos. Vectorconstruction, production of transgenic animals, and verification of thepresence of the transgene in animals were performed generally by themethod of Arbeit et al., 1994, J Virol. 68(7):4358-68, and U.S. Pat. No.5,698,764.

Keratin-14-estrogen receptor (K14-ER) mice exhibited striking changes inthe skin. Specifically, K14-ER mice developed red (erythematous) earsthat were also visibly thickened. Microscopically these ears displayedan increase in the number of cell layers within the epidermis (epidermalhyperplasia), increased synthesis of new DNA, and increased blood vesselformation. This phenotype is stable through at least 2 subsequentgenerations of progeny K14-ER transgenic mice derived from the founder.

Although the foregoing invention is described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to one of ordinary skill in light of theteachings of this invention that a variety of noncritical parameterscould be changed or modified to yield essentially similar resultswithout departing from the spirit or scope of the appended claims.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent specification were specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A transgenic mouse whose genome comprises a humanestrogen receptor gene operably linked to a human keratin-14 promoter,whereby expression of the human estrogen receptor gene in an epithelialcell causes a phenotype selected from the group consisting of epidermalhyperplasia and erythematous ears.
 2. The transgenic mouse of claim 1,wherein said expression of the human estrogen receptor gene causesepidermal hyperplasia.
 3. The transgenic mouse of claim 1, wherein saidexpression of the human estrogen receptor gene causes erythematous ears.4. A method of testing a composition for the ability to modulate theactivity of a human estrogen receptor, the method comprising: providinga transgenic mouse whose genome comprises a human estrogen receptor geneoperably linked to a human keratin-14 promoter which directs expressionof the estrogen receptor in an epithelial cell, whereby expression ofthe human estrogen receptor gene causes a phenotype selected from thegroup consisting of epidermal hyperplasia and erythematous ears;administering the composition to the mouse; and detecting changes in thephenotype, wherein a change in said phenotype indicates that acomposition has modulated the activity of said estrogen receptor.
 5. Themethod of claim 4, wherein expression of the human estrogen receptorgene causes epidermal hyperplasia.
 6. The method of claim 4, whereinexpression of the human estrogen receptor gene causes erythematous ears.7. The method of claim 4, wherein said composition is administeredtopically.
 8. The method of claim 4, wherein said composition isadministered orally.
 9. The method of claim 4, wherein said compositionis administered parenterally.
 10. The method of claim 4, wherein saidchanges are detected by visual inspection.
 11. The method of claim 4,wherein said changes are detected by microscopic observation.